The present invention is directed to polynucleotides encoding the Carnitine Carrier Related Protein-1 (CCRP-1) protein, fragments thereof, and the regulatory regions located at the 5xe2x80x2- and 3xe2x80x2-end of the CCRP-1 gene. The invention also concerns polypeptides encoded by the CCRP-1 gene and fragments thereof. The invention is further directed to methods of making said polynucleotides and polypeptides as well as methods of using the same. The invention also relates to antibodies directed specifically against the above polypeptides and to methods of using said antibodies to purify and detect the above polypeptides and to detect mitochondrion organelles.
The majority of mitochondrial proteins are encoded by nuclear genes, are synthesized on cytosolic ribosomes, and are imported into the mitochondria. Nuclear-encoded proteins which are destined for the mitochondrial matrix typically contain positively-charged amino terminal signal sequences. Import of these preproteins from the cytoplasm requires a multisubunit protein complex in the outer membrane known as the translocase of outer mitochondrial membrane (TOM; previously designated MOM; Pfanner, N. et al., 1996) and at least three inner membrane proteins which comprise the translocase of inner mitochondrial membrane (TIM; previously designated MIM; Pfanner et al, supra). An inside-negative membrane potential across the inner mitochondrial membrane is also required for preprotein import. Preproteins are recognized by surface receptor components of the TOM complex and are translocated through a proteinaceous pore formed by other TOM components. Proteins targeted to the matrix are then recognized by the import machinery of the TIM complex. The import systems of the outer and inner membranes can function independently (Segui-Real, B. et al., 1993). Three TIM proteins have been identified in the yeast Saccharomyces cerevisiae. TIM44 is a hydrophilic protein which is peripherally associated with the inner face of the inner mitochondrial membrane. TIM23 and TIM17 are integral membrane proteins which are thought to comprise the core subunits of the inner membrane translocation channel. (Bomer, U. et al., 1996). Depletion of TIM17 (also known as MIM17, Mpi2, and Sms1; Pfanner et al., supra) causes defects in the import of several mitochondrial proteins (Ryan, K. R. et al., 1994). Furthermore, TIM44, TIM23, and TIM17 proteins are among the few known proteins essential for yeast viability (Maarse, A. C. et al. 1994; Ryan et al., supra).
Fatty acids are activated on the outer mitochondria membrane, whereas they are oxidized in the mitochondria matrix. Long chain acyl CoA molecules do not readily traverse the inner mitochondrial membrane, and so a special transport mechanism is needed. Activated long-chain fatty acids are carried across the inner mitochondrial membrane by carnitine zwitterionic compound formed from lysine. The acyl group is transferred from the sulfur atom of CoA to the hydroxyl group of carnitine to form acyl carnitine. This reaction is catalyzed by carnitine acyltransferase I, which is located on the cytosolic face of the inner mitochondrial membrane. Acyl carnitine is then shuttled across the inner mitochondrial membrane by a translocase. The acyl group is transferred back to CoA on the matrix side of the membrane. This reaction, which is catalyzed by carnitine acyltransferase II, is thermodynamically feasible because the O-acyl link in carnitine has a high group-transfer potential. Finally, carnitine is returned to the cytosolic side by the translocase, in exchange for an incoming acylcarnitine. A defect in the transferase or translocase, or a deficiency of carnitine, might be expected to impair the oxidation of long-chain fatty acids.
Uncoupling proteins, such as UCP-1 (thermogenin), are transmembrane proton-translocating proteins present in the mitochondria of brown adipose tissue, a specialized tissue which functions in heat generation and energy balance (Nicolls, D. G., and Locke, R. M., 1984; Rothwell, N. J. and Stock, M. J. 1979). Mitochondrial oxidation of substrates is accompanied by proton transport out of the mitochondrial matrix, creating a transmembrane proton gradient. Re-entry of protons into the matrix via ATP synthase is coupled to ATP synthesis. However, UCP-1 functions as a transmembrane proton transporter, permitting re-entry of protons into the mitochondrial matrix unaccompanied by ATP synthesis. Environmental exposure to cold evokes neural and hormonal stimulation of brown adipose tissue, which increases UCP mediated proton transport, brown fat metabolic activity, and heat production.
Recent studies with transgenic models indicate that brown fat and UCP-1 have an important role in energy expenditure in rodents. Transgenic mice in which brown adipocyte tissue was ablated by a toxin coupled to the UCP-promoter developed obesity and diabetes (Lowell, B. B., et al., 1993). Obesity in these transgenic animals developed in the absence of hyperphagia, suggesting that the uncoupled mitochondrial respiration of brown fat is an important component of energy expenditure. In a separate transgenic mouse model, ectopic expression of UCP-1 in white adipose tissue of genetically-obese mice led to a significant reduction in body weight and fat stores (Kopecky J., et al. 1995). These studies indicate that activity of UCP-1 is accompanied by energy expenditure and weight loss in rodents. Two other UCP proteins have recently been cloned. The first uncoupling protein-like protein (UCPL) or UCP-2, is expressed in multiple tissues, and is enriched in tissues of the lymphoid lineage (Fleury, C., et al., 1997). The second, UCP-3, is predominantly localized to skeletal muscle (Boss, O., et al., 1997). UCP-3 has been found to be regulated by cold and thyroid hormone (Larkin, S., et al., 1997).
Thermogenic protein activity, such as that found with UCP-1, may be useful in reducing, or preventing the development of excess adipose tissue, such as that found in obesity. Obesity is becoming increasingly prevalent in developed societies. Attempts to reduce food intake, or to decrease hypernutrition, are usually fruitless in the medium term because the weight loss induced by dieting results in both increased appetite and decreased energy expenditure (Leibel et al. 1995). The intensity of physical exercise required to expend enough energy to materially lose adipose mass is too great for many obese people to undertake on a sufficiently frequent basis. Thus, obesity is currently a poorly treatable, chronic, essentially intractable metabolic disorder. In addition obesity carries a serious risk of co-morbities including, Type 2 diabetes, increased cardiac risk, hypertension, atherosclerosis, degenerative arthritis, and increased incidence of complications of surgery involving general anesthesia.
The present invention provides isolated CCRP-1 polynucleotides and polypeptides. One aspect of the invention provides isolated nucleic acid molecules comprising or alternatively consisting of polynucleotides having a nucleotide sequence selected from the group consisting of: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:1; (b) a polynucleotide comprising the nucleotide sequence of the human cDNA contained in the deposited clone; (c) a polynucleotide comprising a portion of the nucleotide sequence of SEQ ID NO:1 coding for a mature CCRP-1 polypeptide; (d) a polynucleotide comprising a nucleotide sequence of the portion of the human cDNA contained in the deposited clone coding for a mature CCRP-1 polypeptide; (e) a polynucleotide comprising a nucleotide sequence coding for the amino acid sequence of the full length polypeptide of SEQ ID NO:2; (f) a polynucleotide comprising a nucleotide sequence coding for an amino acid sequence of a mature polypeptide of SEQ IDNO:2; (g) a polynucleotide comprising a nucleotide sequence coding for an amino acid sequence of a full length CCRP-1 polypeptide encoded by the human cDNA contained in the deposited clone; (h) a polynucleotide comprising a nucleotide sequence coding for an amino acid sequence of a mature CCRP-1 polypeptide encoded by the human cDNA contained in the deposited clone; (i) a polynucleotide comprising a genomic sequence coding for a CCRP-1 polypeptide; (j) a polynucleotide comprising the 5xe2x80x2 transcriptional regulatory region of the CCRP-1 gene; (k) a polynucleotide comprising the 3xe2x80x2 transcriptional regulatory region of the CCRP-1 gene; (l) a polynucleotide comprising the nucleotide sequence of any combination of (i)-(k); (m) a polynucleotide comprising a nucleotide sequence of (a)-(l), wherein the polynucleotide is single stranded, double stranded, or a portion is single stranded and a portion is double stranded; (n) a polyncleotide comprising a nucleotide sequence complementary to any of the single stranded polynucleotides of (m). The invention further provides for fragments of the nucleic acid molecules of (a)-(n) above.
Further embodiments of the invention include isolated nucleic acid molecules that comprise, or alternatively consist of, a polynucleotide having a nucleotide sequence at least 90% identical, and more preferably at least 95%, 96%, 97%, 98% or 99% identical, to any of the nucleotide sequences in (a)-(n) above, or a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide in (a)-(n) above. Additional nucleic acid embodiments of the invention relate to isolated polynucleotides comprising a nucleotide sequence coding for an amino acid sequence of an epitope-bearing portion of a CCRP-1 polypeptide.
The present invention also relates to recombinant vectors, which include the isolated polynucleotides of the present invention, and to host cells recombinant for the polynucleotides of the present invention, as well as to methods of making such vectors and host cells. The present invention further relates to the use of these recombinant vectors and recombinant host cells in the production of CCRP-1 polypeptides.
The invention further provides for isolated CCRP-1 polypeptides comprising an amino acid sequence selected from the group consisting of: (a) the full length amino acid sequence of SEQ ID NO:2; (b) the amino acid sequence of a full length CCRP-1 polypeptide encoded by the human cDNA contained in the deposited clone; (c) an amino acid sequence of the portion of SEQ ID NO:2 representing a mature CCRP-1 polypeptide; (d) an amino acid sequence of a mature CCRP-1 polypeptide encoded by the human cDNA contained in the deposited clone; (e) an amino acid sequence of a signal peptide of SEQ ID NO:2; (f) an amino acid sequence of a signal peptide portion of a CCRP-1 polypeptide encoded by the human cDNA contained in the deposited clone; (g) an amino acid sequence of an epitope-bearing portion of SEQ ID NO:2; (h) an amino acid sequence of an epitope-bearing portion of a CCRP-1 polypeptide encoded by the human cDNA clone contained in the deposited clone. The invention further provides for fragments of the polypeptides of (a)-(h) above, such as those having biological activity or comprising biologically functional domain(s).
The polypeptides of the present invention also include polypeptides having an amino acid sequence with at least 70% similarity, and more preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similarity to those polypeptides described in (a)-(h) above, as well as polypeptides having an amino acid sequence at least 70% identical, more preferably at least75% identical, and still more preferably 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to those polypeptides in (a)-(h) above. The invention further relates to methods of making the polypeptides of the present invention.
The invention further relates to antibodies that specifically bind the CCRP-1 polypeptides of the present invention and to methods for producing such antibodies and fragments thereof.
The invention also provides for methods of detecting the presence of the polynucleotides and polypeptides of present invention in a biological sample. One such method involves assaying for the expression of a CCRP-1 polynucleotide in a sample from an animal. An example of such a method involves the use of the polymerase chain reaction (PCR) to amplify and detect CCRP-1 polynucleotides or Southern and Northern blot hybridization to detect CCRP-1 genomic DNA, cDNA or mRNA. Another example of such a method of detecting one or more CCRP-1 polynucleotides in a biological sample comprises the steps of: (a) contacting the biological sample with one or more polynucleotides of the present invention (which may be individually specified), under conditions such that hybridization occurs, and (b) detecting hybridization of said polynucleotides with one or more CCRP-1 polynucleotides present in the biological sample.
The invention also concerns to biallelic markers of the CCRP-1 gene and the use thereof. The invention is further directed to methods for the screening of substances or molecules that inhibit the expression of CCRP-1, as well as with methods for the screening of substances or molecules that interact with a CCRP-1 polypeptide or that modulate the activity of a CCRP-1 polypeptide (either increase or decrease activity).
The present invention further relates to methods of detecting mitochondria by using antibodies which specifically bind the polypeptides of the present invention or by fusing a polypeptide of the present invention, comprising the CCRP-1 mitochondrial signal peptide sequence, or position thereof to either a heterologous polypeptide that can be used as a label directly (e.g., green fluorescent protein) or to a heterologous polypeptide specifically recognized by an antibody that can be used in an immunodetection assay.
The present invention further relates to methods of delivering heterologous polynucleotides to mitochondria by fusing or linking (covalently or non-covalently) the heterologous polynucleotide of interest to a composition comprising a CCRP-1 mitochondrial signal peptide sequence (xe2x88x9268 to xe2x88x921 of SEQ ID NO:2 or the signal peptide sequence of a polypeptide encoded by human cDNA of the deposited clone) or portions thereof.
The present invention further relates to methods of delivering small molecules, such as bioactive or mitotoxic compounds (e.g., DNP, lipophillic cations), to mitochondria by linking the small molecule to a composition comprising a CCRP-1 mitochondrial signal peptide sequence (xe2x88x9268 to xe2x88x921 of SEQ ID NO:2 or the signal peptide sequence of a polypeptide encoded by human cDNA of the deposited clone) or portions thereof.
The present invention further relates to methods of delivering heterologous polypeptides to mitochondria by fusing the heterologous polypeptide of interest to polypeptides of the present invention comprising the CCRP-1 mitochondrial signal peptide sequence (xe2x88x9268 to xe2x88x921 of SEQ ID NO:2 the signal peptide sequence of a polypeptide encoded by human cDNA of the deposited clone or portions of either).
The present invention further relates to methods of increasing the permeability of mitochondria, thereby causing the nonspecific inhibition of mitochondrial enzymes leading to a decrease in ATP production, and alternatively cell death, comprising administering to in vitro cell cultures or an animal a composition comprising a polypeptide of the present invention, wherein the polypeptide comprises a CCRP-1 mitochondrial signal peptide sequence or portion thereof.
The present invention further relates to insect, bird, plant and mammalian cells with an enhanced ability to metabolize fatty acids, wherein the cells are transiently or stably transfected or transduced with a polynucleotide that expresses a polypeptide of the present invention.
The present invention further relates to a transgenic plant or animal, preferably mammals, fish, and birds, more preferably, a mouse, rat, horse, cow, pig, sheep, chicken, dog, cat, wherein the animal is transgenic for a polynucleotide of the present invention and expresses a polypeptide of the present invention.
The present invention further relates to a method for enhancing a cells ability to metabolize,grow or be maintained under conditions where the fatty acids are present, preferably at levels higher than normal.
The present invention further relates to a device, physiological acceptable composition and method for metabolizing fatty acids in an animal or individual (host) thereby reducing an individual""s blood levels of fatty acids and alternatively, in addition, reducing the level of, or reducing the increase in, white adipose tissue. More particularly, the inventive device is an extracorporeal device for metabolizing fatty acids comprising a semipermeable membrane having a first and a second side and having a molecular weight cutoff of at least 10,000 daltons, an oxidizing component located adjacent to the first side of the semipermeable membrane comprising an enzyme system with necessary cofactors, brown fat mitochondria or whole cell cultures of brown adipose cells of any species or cells transfected with a construct comprising a CCRP-1 polynucleotide sequence alone or combined with a heterologous uncoupling protein (UCP) polynucleotide sequence, referred to hereafter as CCRP-1/UCP, each regulated by an appropriate promoter sequence (e.g., MMTV, SV40, CMV intermediate early, etc.), either combined on a single vector or on separate vectors, wherein the oxidizing component is capable of oxidizing fatty acids, and a means for circulating blood from the host to the second side of the semipermeable membrane for triglyceride hydrolysis and diffusion of free fatty acids to the first side of the semipermeable membrane for oxidation of fatty acids and returning treated blood to the host. Preferably, the oxidizing component comprises a culture of brown fat cells or other eukaryotic cells transfected with a gene encoding a CCRP-1 polypeptide or CCRP-1/UCP polypeptide(s) in an expression vector. Preferably the semipermeable membrane has a lipoprotein lipase embedded therein.
The present invention further provides a physiologically acceptable composition for metabolizing fatty acids comprising a culture of brown fat cells or CCRP-1 or CCRP-1/UCP transfected cells encapsulated in a porous growth matrix and having a semipermeable membrane encapsulating the porous growth matrix. The semipermeable membrane has a molecular weight cutoff of at least 10,000 daltons and, preferably, a lipoprotein lipase embedded therein. Preferably, the semipermeable membrane comprises a tubular membrane having two ends, filled with brown fat cells in the porous growth matrix and sealed at both ends prior to subcutaneous, intramuscularor, or intraperitoneal implantation. Preferably the porous growth matrix comprises alginate beads or another complex polysaccharide porous matrix suitable for cellular growth and metabolism.
The present invention further provides a physiologically acceptable composition for metabolizing fatty acids comprising a mammalian cell stably transfected with a DNA sequence(s) coding for a CCRP-1 or CCRP-1/UCP polypeptides, wherein the transfected mammalian cell transcribes and translates the CCRP-1 or CCRP-1/UCP polypeptides. Preferably, the transfected mammalian cell further comprises a cDNA sequence that confers antibiotic sensitivity to the mammalian cell as a xe2x80x9csuicide genexe2x80x9d mechanism to remove the transformed mammalian cell from an individual if treated with said composition. Most preferably, the antibiotic is gancyclovir.
The present invention further provides a physiologically acceptable composition for metabolizing fatty acids comprising a cDNA sequence encoding a CCRP-1 or CCRP-1/UCP polypeptide(s) in combination with appropriate regulatory and promoter sequences, wherein said cDNA sequence(s) is taken up into hosts cells, in vivo or in vitro, and is translated into CCRP-1 or CCRP-1/UCP polypeptide(s).
The present invention further provides a physiologically acceptable composition for metabolizing fatty acids comprising a culture of allogeneic brown fat cells transfectd or transduced to express a CCRP-1 or CCRP-1/UCP polypeptide(s), wherein the brown fat cells are maintained or proliferated ex vivo.
Further still, the present invention provides a method for maintaining a lower percentage of white adipose tissue than normal or effecting weight loss in a host, wherein the lean state or weight loss is due to prevention of accumulation, or loss, of white adipose tissue, with minimal loss of muscle mass, wherein the method for maintaining a lean state or effecting weight loss comprises administration of an effective amount of a physiologically acceptable composition described herein in sufficient amounts to metabolize at least 25, preferably at least 55 calories or 25 g per day, preferably at least 65 g per day of fatty acids and in some embodiments more than 65 g per day.
The invention further relates to methods of screening and identifying individuals at increased risk for developing certain diseases/disorders, including hyperinsulinemia, glucose intolerance, type II diabetes, obesity, syndrome X, immunological dysfunction and body temperature dysfunction, and heart disease.
The present invention also relates to methods of identifying individuals having elevated or reduced levels of CCRP-1, which individuals are likely to benefit from therapies to suppress or enhance CCRP-1 expression, respectively.
The present invention also relates to methods of screening compounds for their ability to modulate (e.g. increase or inhibit) the activity or expression of CCRP-1. More specifically, the present invention relates to methods of testing compounds for ability either to increase or to decrease expression or activity of CCRP-1.
The present invention also relates to pharmaceutical or physiologically acceptable compositions comprising, an active agent, the polypeptides, polynucleotide or antibodies of the present invention.
The present invention further relates to methods of reducing fatty acid blood levels and treating diseases/disorders such as hyperinsulinemia, glucose intolerance, diabetes, obesity, syndrome X, heart disease, cancer and hypothermia by increasing CCRP-1 activity and/or expression.
The present invention further relates to methods of reducing fatty acid blood levels and treating diseases/disorders such as hyperinsulinemia, glucose intolerance, diabetes, obesity, syndrome X, heart disease, cancer and hypothermia by increasing CCRP-1 activity and/or expression.